1. Field of the Invention
The present invention relates to the field of genetic engineering, and more particularly relates to methods of targeting endogenous and exogenous genes to transgenic animals through homologous recombination.
2. Description of the Related Art
Over the past decade, transgenic technology has developed rapidly and been widely applied to different areas. Combined with cloning technology, it makes possible for bringing transgenic animals from basic research to industrial production. Expressing target genes stably and efficiently in transgenic animals has been a great challenge for its application in industrial production. One important factor affecting the expression efficiency of a target gene is integration sites of target genes in host genomes. Therefore, identification and characterization of sequences associated with high efficiency integration and stable expression of target genes is very important for industrial application of transgenic animals.
The ROSA26 was originally identified as a ubiquitous marker in a retroviral gene-trapping screen in mouse embryonic stem cells. The promoter for ROSA26 gene was found to drive widespread expression of transgenes, and the promoter region and the first intron of the ROSA26 forward transcript has been established as the preferred integration site for the high targeting efficiency and the ubiquitous expression of reporter genes in transgenic mice. There are many transgenic mice reported, such as Zambrowicz et al (Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci USA, 1997, 94(8): 3789-3794), Kisseberth et al (Ubiquitous expression of marker transgenes in mice and rats. Dev Biol, 1999, 214(1): 128-138). In 2007, Trion et al. have documented the identification and characterization of a human homolog of the mouse Rosa26 locus in human embryonic stem cells and demonstrated that transgenes can be readily introduced and broadly expressed in the different lineages of human cells via homologous recombination at the human Rosa26 locus (Trion et al. Identification and targeting of the ROSA26 locus in human embryonic stem cells. Nat Biotechnol, 2005, 25: 1477-1482). Although, the ROSA26 gene has been used widely in transgenic mice, there is no application of ROSA26 gene in transgenic livestock.
The previous studies of transgenic livestock largely rely on random integration of target genes into the livestock genome. This can be problematic as transgenes introduced into random sites with multiple copies are often subjected to position effect variegation (PEV) and repeat-induced gene silencing (RIGS). The method causes the high variability of expression which frequently happens by random integration due to transgenes inserted at random and at various copy numbers into the host genome. In addition, multiple transgene copies and unpredicted integration sites also makes it difficult for identification of transgenic animals, which has been an obstacle for widespread usage of transgenic animals in industrial production. Research shows that targeted transgensis though homologous recombination can provide a solution to the problems above. However, targeted transgensis has only been applied successfully in transgenic mice, fewer in other transgenic livestock, especially no application in transgenic pigs.
There is a need for identifying genomic locus with high homologous combination frequency and ubiquitous transcriptional activity in pigs. The present invention satisfies this need and provides other benefits as well.